The search for disease biomarkers has created a demand for rapid protein analysis from serum, plasma, and other complex protein mixtures. Several separation modalities, including chromatography, electrophoresis, isoelectric-focusing, and mass spectrometry are used in the search for biomarkers for numerous disease states.
Serum and plasma, for example, contain a rich source of biomolecules including a complex mixture of proteins. It is believed that many proteins, including newly synthesized proteins and especially degraded protein fragments, are transported in blood. Because of this, the concentration of newly synthesized proteins or proteins that are being degraded may vary depending on circumstances. This can be especially true in some disease states, thus potentially allowing diagnosis of such diseases through the presence of certain biomarkers. Serum, however, presents challenges for the physical detection of such proteins as they are present at substantially lower concentrations as compared to high abundant proteins. Of the thousands of proteins that are present in blood, only a handful make up a vast majority of the total protein mass in serum. Thus these “high abundant” proteins can cause interference problems with most if not all protein separation methods.
One exemplary method that is often utilized to remove two of the high abundant proteins from serum, namely albumin and IgG, is based on immunoaffinity chromatography. These affinity columns contain immobilized antibodies against human albumin and IgG that function to bind these proteins. The remainder proteins that do not, in theory, bind to the antibodies in the affinity column are retrieved in a pass-through fraction. This method of extraction can be expensive and inefficient, as only small amounts of serum (e.g. less than 50 μl) can be depleted of albumin and IgG on small columns (1 ml or less).